Bottom Line:
The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified.Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking.The gene transfer is efficient and the preliminary safety data are encouraging.

Affiliation: F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACTChoroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.

Mentions:
Two unrelated individuals with clinical diagnoses of choroideremia consented to submit blood samples for the generation of blood-derived cell lines and for molecular diagnoses. These cell lines are available except where this would breach confidentiality rules related to human subject research. For the generation of induced pluripotent stem cell lines (iPSc), peripheral blood mononuclear cells were transduced using the STEMCCA lentiviral vector containing the four Yamanaka reprogramming genes [31]. Cell lines from the two individuals were designated as CPS1 and CPS2, respectively. A schematic for the generation of CPS1 iPSCs is shown in figure 2A. A normal G-banded karyotype is shown in figure 2B. The iPSC lines were characterized for standard quality control criteria such as morphology (figure 2B) surface expressed pluripotency markers such as SSEA-4, and TRA-1-60 (figure 2C) [34], and gene expression of pluripotency markers such as DNMT3B, REX-1, OCT4, and NANOG (figure 2D) [35], [36]. All clones were maintained in culture for a minimum of sixteen passages before analyses to erase residual epigenetic memory associated with the cell of origin [32], [37]. The pluripotency ability of CPS clones was assessed by teratoma formation assay, generating each germ layer (ectoderm, mesoderm, and endoderm) (figure 2E).

Mentions:
Two unrelated individuals with clinical diagnoses of choroideremia consented to submit blood samples for the generation of blood-derived cell lines and for molecular diagnoses. These cell lines are available except where this would breach confidentiality rules related to human subject research. For the generation of induced pluripotent stem cell lines (iPSc), peripheral blood mononuclear cells were transduced using the STEMCCA lentiviral vector containing the four Yamanaka reprogramming genes [31]. Cell lines from the two individuals were designated as CPS1 and CPS2, respectively. A schematic for the generation of CPS1 iPSCs is shown in figure 2A. A normal G-banded karyotype is shown in figure 2B. The iPSC lines were characterized for standard quality control criteria such as morphology (figure 2B) surface expressed pluripotency markers such as SSEA-4, and TRA-1-60 (figure 2C) [34], and gene expression of pluripotency markers such as DNMT3B, REX-1, OCT4, and NANOG (figure 2D) [35], [36]. All clones were maintained in culture for a minimum of sixteen passages before analyses to erase residual epigenetic memory associated with the cell of origin [32], [37]. The pluripotency ability of CPS clones was assessed by teratoma formation assay, generating each germ layer (ectoderm, mesoderm, and endoderm) (figure 2E).

Bottom Line:
The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified.Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking.The gene transfer is efficient and the preliminary safety data are encouraging.

Affiliation:
F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACTChoroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.